CBFA1 is a transcription factor with the most well established role in osteoblast differentiation and biomineralization. However, there are hardly any known CBFA1 target genes, which seem to play the anticipated critical role in promoting biomineralization. No study has been reported to date, describing an unbiased pursuit of CBFA1 target genes. This application proposes to clone new CBFA1 targets in osteoblasts, with the long-term goal of discovering genes playing critical roles in biomineralization. Traditional approaches to discover CBFA1 targets (e.g., differential display, microarrays) would compare mRNAs from cells with normal, low, or high levels of CBFA 1. However, such approaches often result in a long list of genes, for many of which the altered expression is secondary and of little importance to the transcription factor and to the biological process of interest. In addition, these approaches would normally entail over-expression of CBFA1 to supra-physiological levels, possibly leading to the identification of targets with limited biological significance. We have begun to develop a novel approach, by which CBFA1 target genes would be cloned based on their physical interaction with CBFA1 in living osteoblasts. Then CBFA1 targets will be isolated from a pool of genomic DNA fragments obtained by chromatin immunoprecipitation (ChIP) with CBFA1 antibodies. Our preliminary experiments show that, using the most optimal ChIP conditions, known CBFA1 targets are enriched by only 50-fold. By itself, this would be insufficient for isolating CBFA1 targets, due to vast excess of fragments that would non-specifically coprecipitate along with true CBFA1 targets. Towards establishing a method of purifying true CBFA1 targets, we first showed that efficient ChIP could be performed using restriction enzyme digestion instead of sonication for fragmentation of the chromatin. This will facilitate the concentration of CBFA1 target genes, each now represented by a unique size fragment, using polyacrylamide gel electrophoresis. Non-specifically precipitated, contaminating fragments will be spread along a much larger area of the gel. Identification of true CBFA1 targets will be further facilitated by segregating the ChiPped DNA into families of fragments based on the identity of nucleotides at the ends of each fragment. This step, achieved by amplification of the Chipped fragments with pairs of anchored primers, will further increase the intensity of bands representing true CBFA1 fragments over the background. Finally, the most intense bands will be eluted from the gel, cloned and sequenced. Genetically adjacent ORFs will be identified and their regulation by CBFA1 validated.